1. Field of the Invention
The present invention relates to Saxatilin, a protein derived from a Korean snake, Agkistrodon saxatilis emelianov, a process for preparing Saxatilin, and pharmaceutical application of the same, more specifically, to Saxatilin, a protein derived from the venom of a Korean snake, Agkistrodon saxatilis emelianov, a process for preparing Saxatilin, and pharmaceutical application of the same as anti-platelet aggregation agent and anti-tumor agent.
2. Description of the Prior Art
Tumor invasion and metastasis are defined as the proliferating stages of the tumor cells at whole body from the primary tumor, which finally persuade the patient into death. Cancer cells detached from the primary malignant neoplasm (mostly endothelium) penetrate the basement membrane which separates the cancer cells from the other tissue. As some penetrated cells can invade not only endothelium but also basement membrane which surrounds the blood vessels, they can migrate freely via blood vessels and finally set down on capillary vessels. If cancer cells penetrate capillary vessels again, they can form a secondary tumor. The probability of secondary tumor formation from the primary tumor followed by dissemination and invasion is under one in ten thousand (see: Erkki R., Scientific American, 257:72–77 (1996)).
The interaction of cell and extracellular matrix (ECM) is required for the tumor invasion and metastasis. During metastasis, tumor cells induce endothelial cell death, leading to exposure of basement membrane, which facilitates adhesion of tumor cells with ECM protein in surrounding stroma (see: Hynes, R. O., Cell, 48:549(1987)). These substrate proteins promote cell adhesion by binding with cell surface receptor including integrin family.
In terms of structure, each integrin is a heterodimer formed by α subunit and β subunit via non-covalent binding. It has been reported that β1 subfamily plays a role in cell-cell interaction directly as well as in cell-ECM substrate adhesion as a major contact mediator (see: Larjava, H. et al., J. Cell. Biol., 110:803–815 (1990)). β2 subfamily distributed in leukocyte includes cell surface receptors which mediate cell-cell interaction. β3 subfamily including vitronectin receptor and thrombocyte glycoprotein IIb/IIIa complex may be able to function in the development of tumor invasiveness and in the progress of tumor into the malignant tumor (see: Albelda, S. M. et al., Cancer Res., 50:6757–6764 (1990)).
The integrin receptor complex that transversely spans the cell membrane plays a role in connecting cytoskeletal network with extracellular matrix. The core sequences, common to cell-adhesion molecules like fibrinogen, vitronectin and laminin are known to be responsible for cell adhesion, spread and integration. By the way, it is also suggested that cancer promotion and metastasis can be closely related to the role of integrin (see: Giancotti, F. G. and Rouslahti, E., Cell, 60:849–859 (1990); Hynes, R. O., Cell, 69:11–25 (1992); Nip, J. et al., J. Clin. Invest., 30 96:2096–2103 (1995)). Over-expression of fibronectin receptor α5β1 is known to diminish a mutated phenotype in CHO (Chinese hamster ovary) and decrease the expression level of integrin α5β1 in the rodents cells which are mutated into ras (see: Plantefaben, L. C. and Hynes, R. O., 35 Cell, 56:281–290(1989)). Super-fibronectin, a fibronectin polymer, has been reported to prevent cancer promotion and metastasis (see: Pasqualini, R. et al., Nature Medicine, 2:1197–1203 (1996)).
Integrin αVβ3 can be useful as a marker of most malignant tumor cells, which shows the function of integrin in the development of malignant human melanoma (see: Albelda, S. M. et al., Cancer Res., 50:6757–6764(1990)). The expression and adhesive phenotype of integrin αV gene have a direct correlation with in vivo proliferation of human melanoma (see: Felding-Habermann, J. Clin. Invest., 89:2018–2022 (1992)).
Meanwhile, angiogenesis is the process of new blood vessels formation by sprouting of new vessels from existing blood vessels (see: Folkman, J. and D'Amore, P. A., Cell, 87:1153–1155 (1996)). Angiogenesis occurs during development, wound healing and inflammation, and is the essential process in the tumor growth. Cell-adhesion molecules regulate angiogenesis in smooth muscle cells and capillary endothelial cells (see: Nguyen, M. et al., Nature, 365:267 (1993)).
The turnover of tumor angiogenesis is determined by the balance change between stimulating and inhibiting regulators. In relation to this, two cytokine-dependent pathways of angiogenesis have existed and are distinguished by the distinct vascular cell integrins, αVβ3 and αVβ5. These two integrins are expressed in newly formed blood vessels and play an important role in angiogenesis stimulated by bFGF (basic fibroblast growth factor), TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor) and fragments of human tumors (see: Friedlander, M. et al., Science, 270:1500–1502 (1995)). The activation of αVβ3 integrin stimulates a survival signal inducing the vessel proliferation and differentiation, which demonstrates that the signal transmission by cytokine and integrin receptor is related to angiogenesis (see: Brooks, P. C. et al., Cell, 79:1157–1164 (1994)).
Disintegrins, which are known to be potent antagonists of integrin, are small proteins derived from snake venom (see: Niewiarowski, S. et al., Semin. Hematol., 31:289–300(1994)). Most of disintegrins contain Arg-Gly-Asp (GRD) or Lys-Gly-Asp (KGD) motifs recognized by thrombocyte fibrinogen receptor, α2bβ3. It is reported that disintegrins containing RGD sequences inhibit adhesion of integrin-mediated metastatic cells with ECM and finally block metastasis (see: Trikha, M. et al., Cancer Res., 54 (8):4993–4998(1994)). Integrin αVβ3 has been identified as a marker of angiogenic vessels in chicken embryo and human cells (see: Brooks, P. C. et al., Science, 264:569–571 10(1994)). Monoclonal antibody of integrin αVβ3 disrupts angiogenesis by inducing apoptosis of the newly formed vascular endothelial cells. Synthesized peptides containing RGD sequence, which is known to prevent integrin αVβ3 from binding with ligands, inhibit tumor-induced angiogenesis (see: Brooks, P. C. et al., Cell, 79:1157–1164(1994)) of CAM (chick chorioallantoic membrane). Furthermore, Angiogenin, which is known to help adhesion and proliferation of endothelial cell as a subsidiary factor, is also inhibited by synthesized RGD peptides. Recently, the snake venom-derived disintegrin, Triflavin was reported to inhibit TNF-α stimulated angiogenesis. These results provide the possibility that disintegrins, synthesized RGD peptides and anti-αVβ3 monoclonal antibodies, may be developed as anti-cancer drugs.
On the other hand, the snake venom is known to contain various kinds of proteins that affect thrombosis and hemostasis. Since thrombus formation resulted from the abnormal platelet coagulation raises lethal thrombosis, platelet aggregation antagonist or agonist was isolated from the snake venom and identified. As for aggregation mechanism, platelet aggregation is mediated by fibrinogen binding with a glycoprotein in the platelet of GP IIb/IIIa receptor, where the amino acid sequence of Arg-Gly-Asp (RGD) of fibrinogen binding site has been reported to be crucial for fibrinogen binding with GP IIb/IIIa receptor (see: Rouslagti E. and Pierschbacher M. D., Science, 238:491–497 (1987)). Therefore, proteins containing the said sequence competitively inhibit fibrinogen binding with GP IIb/IIIa receptor which leads to the suppression of platelet aggregation. At first, inhibitors of GP IIb/IIIa receptor isolated from the snake venom were small proteins of 5 kD to 9 kD (see: Huang, T. F. et al., J. Biol. Chem., 262:16157–16163 (1987)). Afterwards, various kinds of platelet aggregation inhibitors, which included large molecular weight antagonists, were isolated from the snake venom. These inhibitors are rich in cysteins, and common in binding with GP IIb/IIIa receptor by the interaction with the Arg-Gly-Asp (RGD) amino acid sequence. In addition, the structures of Kistrin (see: Alder, M. et al., Science, 253:445–448 (1991); Alder, M. et al., Biochemistry, 32: 282–289 (1993)), Flavoridine (see: Klaus, W. et al., J. Mol. Biol., 232:897–906 (1993); Senn, H. and Klaus, W., J. Mol. Biol., 232:907–925 (1993)), Albolabrin (see: Jaseja, M. et al., Eur. J. Biochem., 218: 853–860 (1993)) and Echistatin (see: Chen, Y. et al., Biochemistry, 30:11625–11636 (1991); Cooke, R. M. et al., Eur. J. Biochem., 202:323–328 (1991); Cooke, R. M. et al., Protein Eng., 5:473–477 (1992); Saudek, V. et al., Biochemistry, 30:7369–7372 (1991); Dalvit, C. et al., Eur. J. Biochem., 202:315–321 (1991)) have been identified by nuclear magnetic resonance (NMR) spectroscopy. And, the inhibition of fibrinogen binding with GP IIb/IIIa receptor by these antagonists was ascertained by using animal model (see: Collen, B. S., J. Clin. Invest., 76:101–108 (1985); Gold, H. K. et al., Circulation, 77:670–677 (1988); Yasuda, T. et al., J. Clin. Invest., 81:1284–1291 (1988); Coller, B. S. et al., Blood, 68:783–786 (1986); Hanson, S. R. et al., J. Clin. Invest., 81:149–158 (1988)).
In relation to this, extensive studies have been made on the venom of Agkistrodon halys brevicaudus or Caliginosus. Salmosin derived from the venom of Agkistrodon halys brevicaudus, a protein of about 7.5 kD, has been proposed as a potent platelet aggregation inhibitor (see: Korean Patent No. 142606). However, little is known about the venom of Agkistrodon saxatilis emelianov because of the rareness of Agkistrodon saxatilis emelianov compared with other snakes. This invention was made on the assumption that the potent toxicity and high lethality of Agkistrodon saxatilis emelianov venom may be closely related with the bleeding.